Tubulin tyrosinolation in neutrophils is stimulated by the chemotactic peptide fmet-leu-phe and the calcium ionophore A23187. This post-translational reaction requires calcium and activation of the NADPH oxidase. Neutrophil cytoplasts fail to demonstrate tubulin tyrosinolation in response to these stimuli indicating a requirement for intact neutrophils. Use of phorbol myristate acetate to activate neutrophils results in tyrosine incorporation into multiple proteins and is independent of protein synthesis. The phenomenon is dependent upon NADPH oxidase activation and is absent in neutrophils form patients with chronic granulomatous disease. Multi-protein tyrosinolation did occur in myeloperoxidase deficient neutrophils exposed to phorbol myristate acetate. The phorbol esters stimulate a two-fold increase in generation of protein carbonyl derivatives which is potentiated in the presence of labeled tyrosine. The phenomenon is specific for tyrosine as other amino acids like phenylalanine, leucine, histidine or methionine, fail to incorporate. The biochemical mechanism and the functional role of this intriguing reaction remains to be elucidated. However, the data suggest that tyrosine cross linking of peptides may be an important consequence of cell activation.